ABSTRACT
Background: SARS-CoV-2 infects the host by binding of its spike receptor binding domain (S RBD) to angiotensin converting enzyme 2 (ACE2) on the surface of cells in the respiratory tract and gut;thus therapeutics that prevent this interaction are of interest because they have the potential to inhibit propagation of infection. A recombinant 'ACE2 Decoy' that competitively binds S RBD and neutralizes SARS-CoV-2 infection represents such a therapeutic approach. The impact of S RBD mutations found in the rapidly spreading UK, South African, and California SARS-CoV-2 variants on ACE2 Decoy affinity is an important factor in design. Here, we identified a high-affinity SARS-CoV-2-neutralizing ACE2 Decoy that maintains its high affinity against RBD with mutations found in emerging variants. Methods: We used Molecular Dynamic (MD) simulation of S RBD-ACE2 interactions to predict ACE2 residues that if mutated, might increase affinity for S RBD and thus successfully compete with endogenous ACE2 for binding. Recombinant ACE2-IgG1Fc and-IgAFc fusion proteins expressing mutations predicted to increase S RBD binding affinity were produced, purified, and screened for binding affinities against wild type S RBD and S RBD expressing E484K, K417N, N501Y, or L452R alone and in combinations of: K417N/N501Y;E484K/N501Y;K417N/E484K;and K417N/E484K/N501Y (found in the South African variant). The ability of the Decoy with the highest affinity to neutralize SARS-CoV-2 infection was determined by a live virus assay using Vero E6 cells. An additional mutation (H374N) to inhibit enzymatic activity of ACE2 was added to the Decoy with the highest affinity from mutation screening. Results: Eleven of the ACE2 mutations predicted to increase affinity for S RBD were tested, revealing the ACE2 Decoy with T27Y and H34A mutations to have the highest S RBD affinity and the ability to neutralize SARS-CoV-2 infection of cells. Both the ACE2 wild type (WT)-IgG1Fc and the ACE2(T27Y/H34A/H374N)-IgG1Fc Decoys showed enhanced binding affinity for S RBD with either N501Y and L452R mutations alone. Interestingly, the South African variant K417N mutation alone decreased affinity, but not in combination with the N501Y mutation. Conclusion: The potential efficacy of the ACE2 (T27Y/H34A/H374N)-IgG1FC Decoy is not decreased by the UK, South African, or California variant mutations and will undergo testing in animal models likely by expression using a human adenovirus (hAd5) construct to enhance stability.